In the medical field it is important to be able to determine whether or not normally sterile bodily fluids, such as blood, contain microorganisms. The presence of microorganisms in blood samples is indicative of a serious infection and can be life-threatening if not detected in a timely fashion.
There are several methods in current use to detect microorganisms. In manual systems, a sample of material to be tested is incubated, usually in a suitable growth medium. Various manipulations such as agitation are required during the incubation and monitoring period. The detection of growth is achieved by visual inspection. For example, technicians observe and assess the growth of bacteria on a Petri dish, or evaluate the clarity of a broth (turbidity). The visual observations and assessments are subjective and, therefore, subject to error. In addition, these manual methods are labor intensive, require significant manipulation, and entail observation of all samples by laboratory personnel.
A number of methods have been suggested to detect the presence or absence of microorganisms by less subjective means. U.S. Pat. No. 3,743,581 (1973), Cady et al, discloses a method for monitoring microbiological growth by measuring the change in the conductivity of selected nutrient media inoculated with a sample. This method reduces inaccuracies arising from human observation of organism growth. However, the method is relatively costly. In addition, it is relatively complex because temperature variations and movement or turbulence adversely affect this method. This is a disadvantage because optimal growth rates of microorganisms occur by shaking or agitating the cultures. Inasmuch as this method relies on conductivity, the media and sample must be stationary during measurement. On the other hand, optimal growth may occur when the cultures are agitated, known in the art as shaking cultures, which is done in an appropriate apparatus for shaking the container.
U.S. Pat. No. 3,907,646 (1975), Wilkins et al, describes measurement of gas production of microorganisms. A pressure transducer is applied to a test tube and connected to a power source and strip recorders. Measurements are recorded on the strip recorders producing a plot of an electrical signal, which is generated over time, indicative of the presence and quantity of microorganisms. The instrument is very large and cumbersome, making it impractical to monitor multiple samples.
In European Patent No. 0124193, the method for observing the growth of bacteria in a sample includes introducing a sample into a growth medium in a closed vessel, incubating the sample for a period of time, namely 18 hours, causing a change in pressure to force liquid through a needle into a chamber where the change in liquid level can be observed. The volume change can be observed in a number of different ways, including using a simple syringe to give a visual indication of a volume change in the sample of liquid without the need for any electronic equipment (see page 2, lines 5-8). A volume change can also be observed by visually detecting the volume change of liquid in a coiled tube connected to the hypodermic needle. In all the alternative embodiments described in the '193 patent, the needle of a syringe is caused to pierce the closure of the bottle to a level below the liquid in the bottle, whereby a pressure increase forces liquid into a cylinder of the volume indicating device. This is an indirect method to monitor a volume change because the volume change in the head space exerts a force which must be transmitted to the liquid causing the liquid to rise up into the needle. In Example I, the monitoring of volume change in liquid level is applied to a transducer. If the sample is shaken or agitated during the test, as is conventional, the gas in the headspace could pass through the needle above the liquid level and not produce a signal. If there were ambient temperature or pressure changes, the liquid level would change. Thus, in order to know if there has been any such change, the operator must continuously visually monitor the test, which is very impractical. Moreover, the operator cannot be certain what has caused a change, namely a temperature, atmospheric pressure, or growth of microorganisms. Therefore, a false reading may result.
U.S. Pat. No. 4,152,213 describes a system by which the growth of microorganisms in a sealed container is detected by measuring reductions in headspace pressure as the microorganism consumes oxygen and comparing the reduction in pressure to a reference standard of the initial pressure. A vacuum sensor senses a reduction in pressure in the headspace of a container and provides an electrical signal to remote electronics. A major problem with such a system is that it is limited to those organisms that consume oxygen. Many microorganisms do not consume oxygen. Thus, the presence of a vacuum is not a universal indicator of microbial growth. Another problem with such a system is that in many instances the maximum decrease in the headspace pressure is small in comparison to the natural variations of the atmospheric pressure. In addition, this method requires precise pressure sensors since it functions on the basis of absolute value of initial and threshold pressures.
Among the objectives of the present invention are to provide a method and apparatus for detecting the presence of microorganisms in bodily fluids that directly monitor the rate of change in the pressure in the head space above a sample to provide an indication of the microbial growth in the sample; that distinguish and correct for changes in atmospheric pressure; that detect both microorganisms which grow and produce gas or consume oxygen, such that both oxygen consuming and non-oxygen consuming microbial growth can be detected; that detect growth in both microorganisms which have rapid growth and microorganisms which have slow growth; that utilize electronics to detect such mechanisms; that are embodied in a self-contained and simple integral device that fits onto the neck of a bottle or container holding the sample; wherein the self-contained device comprises a disposable fitment and sleeve which covers the cap of the sample bottle and a hypodermic needle; which includes an electronic device including a sensor and electronics removably fitted on the disposable fitment.
In accordance with one aspect of the invention, the method of detecting microorganisms comprises the following steps:
1. Providing a sterile vial or bottle which is filled with a sterile liquid culture medium, the culture medium containing a combination of nutrients particularly adapted for use in detecting aerobic microorganisms or anaerobic microorganisms, as the case may be. The sterile vial is closed by means of an inert gas impermeable closure stopper, such as a rubber closure. The manufacturing process for the nutrient media produces a vacuum within the bottle. This vacuum allows for directly drawing of a specimen, such as a blood sample from a patient.
2. Inserting the bodily fluid, which is to be subjected to the detection of microorganisms, into the vial utilizing a hypodermic syringe or similar device forced through the rubber stopper.
3. Positioning a disposal fitment that includes a collar and a needle over the vial while forcing the hypodermic needle of the device through the closure so that the top of the needle is in the headspace above the liquid culture medium.
4. Positioning an electronic device on the fitment and activating the electronics to periodically monitor the rate of change of pressure which may have occurred due to the presence of microorganisms. If the pressure variation exceeds a predetermined rate of change of pressure due to growth of microorganisms, a signal or alarm is activated. A microprocessor analyzes the absolute value of the rate of change of pressure and compares the rates of change of pressure successively with stored rates of change representing aerobic organisms and anaerobic organisms which have differing rates of change of pressure and either are gas producing and gas consuming, and determines whether the rates of change represent organism growth or extraneous changes such as temperature or atmospheric pressure.
Another object of the present invention is to provide a method and apparatus for detecting microbiological growth in a sealed sample or test container that are adapted for employment in conjunction with monitoring either a single sample container or a multiplicity of sample containers, that are amenable to microprocessor-based implementation for enhanced economy, reliability and reduced size, that are adapted for use in detecting growth of both aerobic and anaerobic microbes, that readily accommodate both microbes that produce relatively rapid change of container headspace pressure and microbes that change headspace pressure more slowly, that may be employed by relatively unskilled operators with little or no operator intervention, and that are adapted in a single implementation for detecting growth of microbes of a variety of types and growth rates.
In such a method and apparatus for detecting microbiological growth in a sealed sample container in accordance with one presently preferred implementation of the invention, pressure within the headspace of the container is monitored for detecting rate of change of such headspace pressure. Presence of microbiological growth within the container is indicated as a function of rate of change of headspace pressure. The rate of pressure change is compared with a standard rate for a family of microorganisms, and growth is indicated when the absolute value of the rate of pressure change (which may be positive or negative) exceeds the absolute value of the standard rate (which also may be positive or negative). For detecting microbiological growths which may have differing growth rates, the absolute value of rate of pressure change is compared to a plurality of standard rates for differing families of microorganisms. At least some of the rates may be positive, corresponding to growth of gas-producing organisms, and at least some of the rates may be negative, corresponding to growth of gas-consuming organisms. In either case, microbiological growth in the test sample is indicated when absolute mathematical value of the rate of headspace pressure change exceeds absolute mathematical value of any of the standard rates.
In the preferred embodiments of the invention, headspace pressure is monitored by positioning a pressure sensor so as to develop an electrical pressure signal that varies as a function of headspace pressure within the container. The pressure signal is sampled and stored at preselected periodic time intervals, and successive signals are compared to each other to determine rate of pressure change. Preferably, each sampled pressure signal is compared to a pressure signal sampled and stored a predetermined number of time intervals previously. In this way, gradual rates of pressure change are detected. Most preferably, each sampled signal is compared to at least two signals sampled and stored differing predetermined numbers of intervals previously so as to determine rates of pressure change over differing time intervals.
The detection method and apparatus so described possess a number of significant advantages over prior art techniques. For example, use of rate of absolute value of the pressure change rather than absolute value of container pressure helps eliminate false growth indications due to changes in ambient pressure, temperature or other like conditions. In the same way, comparison of rate of actual pressure change to one or more predetermined standard growth rates helps eliminate false or erroneous growth indications, while at the same time rendering the invention amenable to detection of growth at differing rates, and to detection of growth of both aerobic and anaerobic microbes. In the same way, determination of rate of absolute value of the pressure change by comparison of current pressure with pressure at differing preceding times helps accommodate differing growth rates in both aerobic and anaerobic microbes.